The overall goal is to obtain a molecular understanding of how vertebrate retinal rods generate an electrical signal that is triggered by light, recovers in darkness and adapts during steady illumination. The principal steps in phototransduction have been identified but the underlying mechanisms are not known in detail. An in-depth understanding of the transduction process is necessary in order to identify molecular components that may be defective in hereditary photoreceptor diseases that cause blindness. While expected advances in molecular medicine may offer viable treatments, their effective use will require extensive knowledge of the molecular elements involved and the functional roles they play in visual transduction. The aim of the proposed research is to provide this kind of information by using the dialyzed detached rod outer segment (ROS) to assay the functional effects of intracellular incorporation of exogenous proteins, peptides and compounds that activate or inhibit selected enzymes in the transaction cascade. When internally perfused under whole-cell voltage clamp with solutions containing ATP and GTP, detached ROS generate electrical light responses having the same sensitivity, kinetics and adaptational properties as intact rods. The events that turn on the electrical response are generally accepted. Light activates a G protein-coupled enzyme cascade that increases the hydrolysis of cGMP causing cGMP-gated cation channels in the ROS surface membrane to close. The molecular events responsible for the recovery of the light response are less well understood and are the primary focus of the present grant which will examine three aspects of the recovery process. One is the inactivation of the light-activated intermediates in the transaction cascade; two is the resynthesis of cGMP that was hydrolyzed by light-activated phosphodiesterase; three is calcium. The closure of cGMP-gated channels leads to a fall in intracellular Ca2+ which regulates the lifetime of various transduction intermediates, stimulates the resynthesis of cGMP and has been postulated to act as the "adaptation signal". Electrical recording will be combined with simultaneous optical measurements using a fluorescent Ca2+ indicator to monitor light-evoked changes in internal Ca2+ in dark and light-adapted ROS.